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Illuminating neural circuits and behaviour
in Caenorhabditis elegans with
optogenetics
Christopher Fang-Yen1,2, Mark J. Alkema3 and Aravinthan D. T. Samuel4
Review
Cite this article: Fang-Yen C, Alkema MJ,
Samuel ADT. 2015 Illuminating neural circuits
and behaviour in Caenorhabditis elegans with
optogenetics. Phil. Trans. R. Soc. B 370:
20140212.
http://dx.doi.org/10.1098/rstb.2014.0212
Accepted: 16 June 2015
One contribution of 15 to a theme issue
‘Controlling brain activity to alter perception,
behaviour and society’.
1
Department of Bioengineering, School of Engineering and Applied Science, and 2Department of Neuroscience,
Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
3
Department of Neurobiology, University of Massachusetts Medical School, Worcester, MA 01655, USA
4
Department of Physics and Center for Brain Science, Harvard University, Cambridge, MA 02138, USA
The development of optogenetics, a family of methods for using light to control neural activity via light-sensitive proteins, has provided a powerful new
set of tools for neurobiology. These techniques have been particularly fruitful
for dissecting neural circuits and behaviour in the compact and transparent roundworm Caenorhabditis elegans. Researchers have used optogenetic
reagents to manipulate numerous excitable cell types in the worm, from
sensory neurons, to interneurons, to motor neurons and muscles. Here,
we show how optogenetics applied to this transparent roundworm has
contributed to our understanding of neural circuits.
1. Introduction
Subject Areas:
neuroscience
Keywords:
Caenorhabditis elegans, optogenetics,
neural circuits
Author for correspondence:
Christopher Fang-Yen
e-mail: [email protected]
One of the fundamental goals of neuroscience is to understand how neural
circuits create behaviour. Caenorhabditis elegans, a 1 mm-long roundworm, possesses a number of unique advantages as a model for addressing this question.
It has a compact nervous system containing 302 neurons (in the adult hermaphrodite) and is the only animal for which the complete ‘wiring diagram’ or
‘connectome’—the complete atlas of synaptic connectivity—has been mapped
[1]. This wiring, and the overall anatomy of the nervous system, are highly
conserved between individual C. elegans. Despite its relative anatomical simplicity, the worm’s neurochemistry and genetics are remarkably similar to those
of mammals. Its nervous system signals in part through the classical neurotransmitters glutamate, acetylcholine, gamma-aminobutyric acid (GABA) and
biogenic amines, along with a large number of neuropeptides.
One of its most important assets of C. elegans as a model has been its optical
transparency. Using differential interference contrast microscopy, Sulston et al.
traced the organism’s complete cell lineage during development [2]. Transparency enabled the targeted killing of specific cells in intact worms using a laser
microbeam coupled through a microscope; this approach has generated a great
deal of our knowledge of C. elegans nervous system function [3]. The worm was
the first animal in which the green fluorescent protein (GFP) was expressed, helping to kindle a revolution in live biological imaging [4]. More recently, early work
in monitoring neural and muscle activity through fluorescence of calcium reporters was performed in worms [5]. In 2005, worms became one of the first animals
in which optogenetic manipulation of excitable cells was performed [6], and they
continue to be important for the development and testing of new opsins.
In this review, we aim to summarize the insights gained about neural
circuits from one decade of optogenetics in worms.
2. Optogenetic methods
(a) Optogenetics strategies
The earliest approaches for genetically conferring light sensitivity to cells were
based on components of the Drosophila visual phototransduction machinery
& 2015 The Author(s) Published by the Royal Society. All rights reserved.
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ChR2 consists of the apoprotein channelopsin-2 (chop-2)
bound to the cofactor retinal, a form of vitamin A, which isomerizes from all-trans to 11-cis upon illumination. In addition
to acting as the chromophore, retinal also stabilizes the ChR2
complex with respect to degradation [14]. In some organisms,
including mice, sufficient endogenous retinal is available,
given a standard laboratory diet, for ChR2 to function. In
C. elegans, retinal must be added to the E. coli food bacteria.
This requirement for retinal supplementation provides a convenient way to control for an animal’s intrinsic sensitivity to
illumination: researchers can compare optogenetic experiments to otherwise equivalent ones performed in the
absence of retinal. In addition, researchers can alter retinal
concentration to manipulate the level of opsin activity.
Several laboratories have developed ChR2 variants with
altered kinetics, conductance, optical spectra and other properties. When illuminated with blue light, wild-type ChR2
shows a large decrease in current within about 7 ms. The
gain-of-function mutant ChR2(H134R) exhibits reduced inactivation and therefore higher steady-state currents relative to
ChR2(wt) [6] and is thus the most commonly used ChR2
variant in C. elegans.
Upon cessation of illumination, wild-type ChR2 deactivates
within about 20 ms. In some applications, a longer period
of activation may be desired. The variants ChR2(C128X) deactivate with time constants varying between 2 and 100 s,
depending on the specific mutation [15]. Owing to their
slow deactivation, these opsins are known as step function
opsins (SFO).
In order to enable differential optogenetic stimulation of distinct cell populations, to monitor intracellular calcium activity in
the presence or the absence of stimulation, or to intrinsic effects
of blue-light stimulation [16,17], researchers have developed
opsins with red-shifted activation spectra. These include the
chimaeric channelrhodopsin C1V1 [18], VChR1 from the alga
Volvox carteri [19], Chrimson [20] and many others [20].
Red-shifted opsins also have the advantage of improved tissue
penetration owing to reduced endogenous absorption.
(c) Opsins for inhibition
After the development of ChR2 for optical stimulation, complementary strategies for optical inhibition rapidly followed.
The first of these was NpHR/Halo, a light-powered chloride
pump from the archaebacterium Natronobacterium pharaonis
(d) Spectrally distinct combinations of opsins
The different spectral properties of various opsins make it possible to address different populations of cells in a spectrally
dependent manner. For example, the Deisseroth and Boyden
groups used blue- and green-light excitation of ChR2 and
NpHR/Halo, respectively, to perform excitation and inhibition of body wall muscles in the same worms [21,22]. More
recently, dual excitation bands with very little crosstalk have
been reported using ChR2 and the red-shifted opsin Chrimson
[20], or by combining Chrimson with blue-light-sensitive
CoChR from Chloromonas oogama [29]. Dual inhibition can be
approached by pairing a blue-light-sensitive inhibitory opsin
with a green- or yellow-light-sensitive inhibitory opsin.
(e) Optogenetics using other signalling pathways
Manipulation of membrane potential via light-activated ion
channels and pumps has become nearly synonymous with
the term optogenetics. However, this strategy is only one of
many approaches for using light to change neuronal function.
Here, we briefly review optogenetics methods for perturbing
other signalling pathways.
Lima and Miesenbock developed a ‘phototrigger’ scheme in
Drosophila based on a key-and-lock mechanism. In this method,
researchers express the ionotropic purinoreceptor P2X2 or the
capsaicin receptor TRPV1 in neurons, and add caged versions
of their agonists. Upon illumination by a flash of light, agonists
are activated, and then bind to the receptors, stimulating the
cells [8].
Another class of optogenetic tools uses ligands attached
to photoisomerizable azobenzene tethers, such that specific
2
Phil. Trans. R. Soc. B 370: 20140212
(b) Opsins for stimulation
[21,22]. Improved versions of this opsin addressed limitations
related to trafficking of the opsin to the plasma membrane [23,24]
Chow, Han et al. used a cross-kingdom functional molecular screen to identify a number of light-driven proton
pumps that can mediate neural silencing [25]. These include
yellow/green-light-drivable archaerhodopsin-3 (Arch) from
Halorubrum sodomense and blue/green-light-drivable Mac
from Leptosphaeria maculans.
ChR2, normally used as an excitatory opsin, can also play
an inhibitory role in two ways. First, in addition to its
capacity as a light-gated cation channel, ChR2 has an outward proton pump activity acting to hyperpolarize the cell
[26]. Under normal conditions the effect on the membrane
potential of this pump activity is small compared with the
cation-mediated depolarization effect.
Second, researchers applying ChR2 in mammalian systems
have reported that repeated stimulation of cells can cause them
to stop firing entirely, and therefore have the effect of inhibiting
rather than exciting them [27,28]. This so-called depolarization
block may result from the kinetics of ChR2 and/or disruption
of ionic gradients during excessive stimuli. Depolarization
block causes a transition between excitatory and inhibitory
effects as a function of ChR2 stimulus time [27] and can
confound interpretation of optogenetics experiments. Susceptibility of cells to depolarization block varies from one cell type
to another and on temperature [28]. While depolarization block
has not yet been demonstrated in C. elegans, it is possible that
it may occur under some conditions. It is therefore important
for researchers to empirically determine the response of
cells, circuits and/or behaviours to the specific stimulation
parameters to ensure that illumination has the intended effect.
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[7,8] or synthetic photoisomerizable groups coupled to receptors [9,10]. Around the same time, Boyden et al. [11] reported
a strategy based on expression of microbial opsins. Heterologous expression of the cation channel Channelrhodopsin-2
(ChR2) from the single-cell alga Chlamydomonas reinhardii [12]
conferred blue-light sensitivity in cultured neurons. The first
in vivo demonstrations of ChR2 function were performed in
chick embryo [13] and in C. elegans body wall muscles [6].
The single-component optogenetic strategy exemplified by
ChR2—expression of microbial ion channels and pumps—is
the most widely adopted by the neuroscience community,
probably owing to its simplicity compared to the multiplecomponent methods. We therefore focus on this approach.
However, researchers continue in efforts to develop strategies
to perturb specific intracellular signalling pathways, as
reviewed below.
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nerve ring
pharynx
head ganglia
3
tail ganglia
ventral nerve cord
Figure 1. Overview of the C. elegans nervous system. The majority of neurons are located in several ganglia near the nerve ring. (Online version in colour.)
(f ) Genetic and spatial targeting
To express opsins in C. elegans, most researchers use standard
methods for transgenesis [33]. The choice of promoter determines the tissues expressing the opsin. In cases where no
single promoter provides adequate specificity, researchers
can apply intersectional promoter schemes based on recombinases FLP or Cre [34,35]. Another method for cell-specific
transgene induction is to stimulate heat shock in single cells
by a pulsed laser beam [36–38].
An alternative to the solely genetic approach to stimulating specific cells is to target the illumination itself to the
desired cells or tissues. Light sources are patterned by a digital micromirror device or liquid crystal display and coupled
onto worms through a microscope. Guo et al. [39] used
such a system to perform simultaneous optogenetic illumination and imaging of neural activity by calcium-sensitive
fluorescent proteins in immobilized worms. Trojanowski
et al. [40,41] used a similar system to perform single-cell excitation and inhibition in the pharyngeal nervous system.
Illumination of selected cells in freely behaving worms
requires computer vision algorithms that infer the position
of targeted neurons based on the outline of the animal.
Several laboratories have used such tracking systems to
target neurons in live behaving C. elegans [42 –44].
(g) Intrinsic light sensitivity
Even without optogenetic manipulation, C. elegans is sensitive
to light in several ways. Researchers should consider these
intrinsic light sensitivities when designing and performing
optogenetics experiments.
In response to illumination with ultraviolet (UV), violet or
blue light, worms accelerate their locomotory rhythms, an
effect mediated by LITE-1, a member of the gustatory receptor
family, and dependent on cAMP and diacylglycerol signalling
[16,17]. This behaviour is thought to help worms avoid potential damage and dehydration owing to sunlight exposure.
Worms have good reason to avoid short-wavelength light:
long-term illumination by bright UV, violet or blue light
causes paralysis and, after approximately 30 min, death [16].
Bhatla & Horvitz [45] showed that bright light inhibits
C. elegans feeding. Like the light-induced locomotory acceleration, this effect is mediated by LITE-1 and also another
gustatory receptor analogue, GUR-3.
To control for intrinsic light sensitivity, experiments
should incorporate control worms lacking retinal and/or lacking the opsin transgene. For cases in which the intrinsic
behaviours cause problems for behavioural assays, researchers
can perform optogenetics experiments in lite-1 mutants defective for endogenous light sensitivity. A recently published
blue-light-sensitive opsin from Chloromonas oogama (CoChR)
has a fivefold higher sensitivity than ChR2 [29]. This allows
CoChR activation at light intensities that do not trigger the
intrinsic light response of C. elegans.
Illumination also causes some degree of warming in worms
and their substrates owing to light absorption. Worms are
highly sensitive to temperature and avoid warming at temperatures higher than that of their recent experience [46].
Experimenters can test whether behavioural responses are
thermal in nature by analysis of thermo-insensitive mutants.
3. Overview of the Caenorhabditis elegans
nervous system
Caenorhabditis elegans has two nearly independent nervous systems: (i) the somatic nervous system containing 282 neurons (in
the adult hermaphrodite) in 118 classes (figure 1) and (ii) the
nervous system of the pharynx (feeding organ), containing
20 neurons in 14 classes [47]. These two nervous systems are
connected by a single electrical synaptic connection between
the somatic RIP neurons and the pharyngeal I1 neurons.
Each C. elegans neuron is identified by a 2- or 3-letter
name and in some cases a number, often followed by one or
more of L (left), R (right), D (dorsal) or V (ventral) to specify
anatomical position.
The worm’s neurons can be classified as sensory neurons,
interneurons and motor neurons, based on their anatomical
features and synaptic connectivity (figure 2). Sensory neurons are those that appear to have sensory endings, whether
or not they have shown to be functional. Motor neurons are
those that make synapses onto muscle cells. Interneurons
are neurons that make many connections with other neurons
[47]. These labels are to some degree arbitrary, since many
neurons span two or more categories. For example, the
B-type excitatory motor neurons form synapses with
several other neuron types and have been shown to have a
proprioceptive sensory function [48].
4. Sensory systems
Optogenetics has played an important role in dissecting the
physiology of C. elegans sensory systems. About 85 of the
Phil. Trans. R. Soc. B 370: 20140212
neurotransmitter receptors can be activated in response to
light [9,10]. This system has the advantage of being able
to target specific receptor types.
Cyclic AMP (cAMP) is a common second messenger in
intracellular signalling; it is a component of G-protein signalling
cascades, and it is involved in synaptic transmission. Photoactivated adenlyate cyclase alpha (PACa) from Euglena gracilis is a
receptor flavoprotein that generates cAMP in response to bluelight illumination [30]. When expressed in C. elegans motor
neurons, PAC enhances neurotransmitter output and causes
worms to increase in speed upon illumination [31]. An infrared-sensitive adenylyl cyclase has also been developed and
shown to be effective in C. elegans motor neurons [32].
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sensory
dendrites
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chemosensory
mechanosensory
nociceptive
gentle touch
AWA
AWB
AWC
ASI
ASJ
ALM
AVM
PVM
PLM
AFD
ASH
soluble
sensory
neurons
harsh touch
ASE
FLP
oxygen
PQR
AQR
URX
AIY
RIA
AIB
AIZ
RIM
RMG
RIB
locomotory
command
interneurons
motor
neurons
head
motor SMD
neurons
RMD
AVB
AVA
PVC
forward
movement
VB
DB
ventral
AVD
AVE
reverse
movement
VNC
motor neurons
VD
muscles
ALA
RIS
interneurons
PVQ
VA
DA
DD
dorsal
Figure 2. Partial circuit diagram of the C. elegans somatic nervous system and musculature. Sensory neurons are represented by triangles, interneurons are
represented by hexagons, motor neurons by circles and muscles by diamonds. Arrows represent connections via chemical synapses, which may be excitatory or
inhibitory. Dashed lines represent connections by electrical synapses. VNC, ventral nerve cord. (Online version in colour.)
hermaphrodite worm’s 302 neurons have been anatomically
classified as sensory cells; some of these have been found to
detect odors, tastes, temperature, mechanical stimuli and light.
Laser killing and genetic experiments have been instrumental
in dissecting sensory modalities in the worm. Optogenetics
has provided a complementary approach to directly test the
behavioural consequences of neuronal activation or inhibition.
Optogenetic activation of sensory neurons that mediate
quick avoidance responses has been particularly robust in recapitulating the endogenous behaviour. Caenorhabditis elegans
exhibits a stereotyped response to non-nociceptive touch to its
body: touch to the anterior part of the body induces backward
locomotion while touch to the posterior part of the body
induces accelerated forward locomotion. Mechanosensation
of gentle touch is mediated by six touch receptor neurons
(TRNs) that extend long processes throughout different regions
of the body [49]. Subsets of the TRNs are necessary to generate
these responses: the anterior touch response requires the
ALM and AVM neurons, while the posterior touch response
requires the PLM neurons. A functional role for PVM has not
yet been demonstrated.
Selective optogenetic illumination of touch cells in freely
moving worms showed that optogenetic activation of individual TRNs elicits avoidance responses indistinguishable from
those observed with touch, demonstrating that depolarization
of the TRNs is sufficient for the induction of avoidance
response [42,43]. Furthermore, optogenetic inhibition of premotor interneurons that drive backward locomotion can
reduce the probability of reversals induced by ALM and
AVM activation [50].
The C. elegans mechanosensory neurons have served as a
model for neuronal regeneration after injury. Axons of touch
cells (and many other neuronal types), after being severed
using a pulsed laser beam, are capable of spontaneous
regrowth [51]. Sun et al. [52] showed that periodic stimulation
by ChR2 can enhance neuronal regeneration after axotomy,
suggesting a novel strategy for beneficial neurotherapy [52].
Analysis of animals deficient in gentle touch response
showed that animals that fail to respond to gentle touch
can still respond to harsh (nociceptive) touch. Laser ablation
studies identified the FLP and PVD neurons as the main sensors of harsh touch [53–56], with additional neurons (SDQ,
BDU, ADE, AQR, PDE, PHA and PHB) contributing to the
response [57]. FLP and PVD are multi-dendritic neurons similar to mammalian nociceptors in both morphology and
function. The analysis of harsh touch response proved difficult since harsh mechanical stimuli also activate the TRNs
that mediate the gentle touch response. Optogenetics allowed
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AIA
DVA
PVD
4
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volatile
thermosensory
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5. Interneurons
In C. elegans, sensory information is transduced into behaviour
by a shallow network of interneurons. Only two or three layers
of interneurons separate sensory neurons from premotor interneurons and motor neurons that regulate motor output. In the
case of mechanosensory avoidance responses, it has been
possible to use laser ablation to establish the feed-forward
pathways that connect sensory stimulation to movement [49].
However, for complex navigational behaviours like chemotaxis
and thermotaxis, it has been challenging to connect sensory
neurons to the regulation of turning movements, forward
movements and backward movements that organize behavioural strategies. Removing specific interneurons from the
circuit may reduce the overall ability to effect chemotaxis and
thermotaxis, but understanding the role of each interneuron
in patterning the sensorimotor transformations that drive
behaviour requires direct physiological analysis.
Optogenetics provides a means of acutely activating or inactivating interneurons during C. elegans navigation, and thus a
means of assigning a role to each interneuron in mediating
specific behavioural outputs. For example, the AVA premotor
interneuron has been shown to be active during backward
movement, and killing AVA reduces the spontaneous reversal
rate during locomotion. Optogenetic activation of AVA can
evoke backward movement. AVA has a number of gap junctions with the RIM interneuron. Guo et al. [39] showed that
activating the RIM interneuron using cell-specific expression
of ChR2 also evokes AVA activity and thereby triggers reversals.
Taken together, these data show that RIM may be part of the circuit for initiating reversals. Interestingly, killing RIM has the
opposite effect to killing AVA, causing animals to have a
lower frequency of spontaneous reversals. One possibility is
that RIM removal from the circuit by laser killing has more
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an increase in excitatory currents as the strength of lightinduced ASH activation increases [39]. This indicates that
the functional connection between ASH and AVA is graded
such that stronger noxious stimuli elicit stronger escape
responses. Similarly, optogenetic activation of the thermosensory AFD neurons combined with recordings of the
downstream interneuron AIY reveal that AFD can tonically
excite or inhibit AIY [70]: inhibition of AFD activity using
halorhodopsin leads to strong activation of AIY, while strong
activation of AFD results in weaker activation of AIY. Therefore, the level of activation of AFD determines whether it
evokes warm-seeking or cold-seeking behaviour [71].
These calcium imaging or electrophysiological experiments
used semi-constrained animals, which may lack proper behaviour feedback mechanisms required for proper neuronal
processing. Novel systems that combine targeted optogenetic stimulation with calcium imaging in unrestrained freely
behaving animals have now been developed that circumvent
this caveat. Using this technique, it was shown that optogenetic
activation of the anterior TRNs induces calcium transients
in the AVA premotor interneurons that correlate with the
worm’s backward velocity. Since the excitation spectra of
ChR2 and GCaMP overlap, this combination can only be
used if the neurons’ bodies are sufficiently far apart. However,
recently developed red-shifted ChR2 or RCaMP (a red
fluorescent calcium reporter) variants with more distinct
excitation spectra [18,19,72] may overcome these limitations.
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the analysis of the harsh touch neurons and downstream circuits without interference by other mechanoreceptor cells.
While photoactivation of the FLP initiated backward movement, activation of the PVD induced forward movement
similar to the endogenous response to harsh touch [50,57].
The optogenetic activation of PVD paved the way for the
identification of the DEG/ENaC and TRP channels required
for its function [50].
The nociceptive ASH neurons detect several noxious stimuli
and are required for avoidance responses to nose touch, high
osmolarity and chemical repellents [58]. Despite the advantages
of targeted illumination in live behaving animals, the spatial
resolution can still be limiting factor when no cell-specific promoters are available and optogenetic proteins are expressed in
cells that are close together. An alternative approach uses FLP
or Cre recombinase and combinatorial expression using promoters whose expression uniquely overlaps in the cell(s) of interest.
Using this approach, it was shown that ASH photoactivation
triggers withdrawal behaviours mimicking the endogenous
response [50,59]. The strength of the optogenetic activation of
the ASH neurons directly correlated with the magnitude
of the behavioural response [50,60].
Sensory neurons required for chemosensation trigger
compound behavioural responses over long time scales that
allow the animal to move up or down chemical gradients.
Caenorhabditis elegans will navigate up or down salt gradients
towards salt concentrations corresponding to concentrations
at which they were raised [61]. The ASE neurons are the
principal sensory neurons that detect salt concentration [62].
ASER is depolarized by decreases in salt concentration,
while ASEL responds to increases in concentration [63,64].
Optogenetic activation of the ASER neuron induced a transient increase in turning for animals on salt concentrations
below the one they were raised at, but a decrease when the
animal navigates above the one they were raised at. Thus,
the ASER neuron mediates salt avoidance and attraction by
regulating the frequency of reorientation movements in
response to the gradient [61,65].
Caenorhabditis elegans prefers oxygen concentrations ranging from 5 to 12% and avoids habitats with low (less than
5%) or high (21%) oxygen concentrations. The principal
oxygen-sensing neurons are the URX, AQR and PQR neurons,
which have sensory endings in the animals’ body fluid [66,67].
Optogenetic activation of the URX, AQR and PQR neurons
increases locomotion, while inhibition of the same neurons
strongly reduces movement, indicating that tonic activation of
the oxygen-sensing neurons stimulates fast movement required
for the escape of unfavourable oxygen concentrations [68].
The primary oxygen sensors, the URX neurons, make gap junction and reciprocal connections with the RMG interneurons.
Light-induced activation of the RMG inhibits spontaneous
reversals and induces rapid forward movement even in animals
in which the URX, AQR and PQR neurons were ablated
[69], indicating that RMG activation is sufficient to induce
an arousal state, probably through the activation of other
downstream interneurons.
The combination of optogenetics with calcium imaging or
electrophysiology in semi-constrained animals has been particularly powerful for unravelling sensorimotor circuits that
mediate behavioural responses to stimuli. For instance, optogenetic activation of ASH induces strong calcium transients in the
AVA and AVD premotor interneurons that drive backward
locomotion. Electrophysiological recordings from AVA show
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6. Motor neurons
The 113 cells classified as motor neurons in the adult hermaphrodite include excitatory cholinergic and inhibitory
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Phil. Trans. R. Soc. B 370: 20140212
Flavell et al. [75] showed that serotonergic neuronal activity
underlies dwelling. Optogenetic activation or inhibition of
serotonergic neurons, including HSN and NSM, upregulated
and downregulated dwelling, respectively. Remarkably,
these effects could also be mimicked by directly manipulating
the activity of serotonergic targets that expressed the MOD-1
serotonin receptor (AIY, RIF and ASI). Optogenetic activation
or inhibition of these downstream targets downregulated
or upregulated dwelling, respectively. Analogous effects
could be produced by optogenetic manipulation of a distinct
circuit that regulates foraging state. The PDF neuropeptide
promotes roaming state, and optogenetic activation of PDF
receptor-expressing cells promotes the roaming state.
During larval development, the worm regularly enters
lethargus, a quiescent sleep-like state during which much of
the nervous system becomes inactive [76,77]. Switching
between quiescence and wakefulness in C. elegans has been
shown to involve the RIA and RIS interneurons that appear
to play opposite roles. Ablation of RIA neurons promotes
quiescence, whereas ablation of RIS disrupts quiescence.
Interestingly, cell-specific optogenetic activation of RIA
during quiescence can activate movement, akin to waking
the worm up [78]. Optogenetic activation of RIS promotes
quiescence [79]. Lethargus can also be interrupted by optogenetic activation of premotor interneurons that evoke
reversals, but this effect requires the synchronous activation
of several interneurons, e.g. AVA, AVD and AVE. Activation
of just AVA does not evoke movement during lethargus [80].
Thus, optogenetic manipulation of a core circuit for quiescence appears to switch the nervous system between a
sleep-like state and wakefulness.
The worm also has distinct behavioural states depending
on ambient oxygen levels. Worms avoid atmospheric oxygen
levels (21%). Exposing worms to high oxygen causes an
increase in speed and reduction in reversal rates that facilitate
escape. The URX oxygen-sensing neurons and their synaptic
partner RMG play a critical role in the entry to the arousal
state upon exposure to high oxygen levels. Laurent et al. [69]
showed that selective optogenetic activation of RMG suffices
to trigger the arousal state. Interestingly, whether the animal
is in the arousal state or not also affects the behavioural effects
of optogenetic activation of other neurons that regulate movement patterns. For example, the AIA interneuron was shown
to have higher calcium levels during forward than backward
movement, whether the animal is exposed to high or low
levels of oxygen. However, optogenetic inhibition of AIA
has distinct effects at high and low oxygen levels. Optogenetic
inhibition of AIA at high oxygen causes an increase in reversal
rate, and optogenetic disinhibition at low oxygen causes a
reduction in reversal rate.
The emerging picture from studies of interneurons using
optogenetics is complex and subtle. The raising and lowering
of activity in any one neuron can have multifarious effects on
the nervous system and behaviour. To be useful, optogenetics
must be judiciously combined with other powerful tools
available in C. elegans including ablation analysis, genetics,
imaging and behavioural analysis.
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complex consequences than RIM inactivation. These results
indicate that optogenetics can yield information that is complementary and distinct from traditional methods of circuit
dissection such as ablation analysis.
In C. elegans, interneurons are highly interconnected with one
another and with downstream circuit pathways. Thus, many
effects might be evoked by optogenetic manipulation of single
neurons. A striking example is the AIY interneuron, which is
downstream of numerous chemosensory and thermosensory
neurons. In a study of olfactory chemotaxis, Kocabas et al. [44]
activated interneurons downstream of the AWC olfactory
neuron with inputs that might resemble those experienced by
an animal trying to navigate towards food. By stimulating or inhibiting the AIY interneuron every time the head
undulated to one side, they showed that it is possible to steer
the animal towards either the dorsal or ventral sides. This
might be related to a ‘klinotactic’ mechanism, by which
the animal augments the curvature of its movements in one direction depending on whether it encounters improving or declining
conditions during a specific phase of its undulation cycle. Similar
results were obtained in a study of salt chemotaxis using phasic
optogenetic stimulation of AIY. Asymmetric activation of any
neuron in a pathway downstream of AIY (AIY ! AIZ ! RIM
! SMB ! RME) also evoked steering [44]. Thus, it is possible
that a cascade of phasic activation of neurons from sensory
neurons to motor neurons generates klinotactic steering.
Interestingly, activating or inactivating AIY using optogenetics can also lower or raise, respectively, the rate of
reversals. The regulation of reversal rate by AIY appears to
require its postsynaptic partner, AIZ. Activating AIY can also
increase the speed of forward movement, but this effect requires
a different postsynaptic partner, RIB [73]. Taken together, these
results suggest that single interneurons in C. elegans can have
multiple roles in behavioural output, roles that are differentiated by the detailed activity pattern of the interneuron and the
downstream pathways that are thereby activated.
Interneurons in C. elegans not only receive input from
upstream sensory layers, but also receive feedback from downstream motor layers. This presents a challenge to interpreting
the behavioural phenotypes that might be evoked by activating
or inactivating individual neurons. The AIB interneuron is
downstream of a number of sensory neurons including the
AWC olfactory neuron. To understand how sensory response
in the interneuron AIB is influenced by the overall network
state, Gordus et al. [74] used optogenetic stimulation in combination with calcium imaging. Optogenetic activation of
AVA, the premotor interneuron for reversals, by the redlight-sensitive cation channel was shown to spread to both
AIB and RIM, influencing the response of these neurons to
olfactory stimulation.
Another complexity in understanding interneuron processing in C. elegans is interpreting the effects of optogenetic
perturbation in the context of behavioural state. A behavioural state of an animal is thought to involve the organized
activity of many brain circuits towards a specific behavioural goal. As described below, it has been shown that
C. elegans’ behavioural state can be modulated by optogenetic
stimulation of specific neurons. It has also been shown
that how optogenetic manipulation modulates behaviour
depends on behavioural state.
Caenorhabditis elegans alternates between two behavioural
states during foraging. During roaming the animal has low
reversal rates, while reversals increase during dwelling.
Downloaded from http://rstb.royalsocietypublishing.org/ on June 17, 2017
(a) Synaptic transmission
Optogenetics has been helpful for understanding the neural
basis of locomotion. Worms move forward by propagating
dorsoventral bending waves from anterior to posterior.
Navigation occurs in part via brief reversals during which
the bending wave proceeds from posterior to anterior.
Motor neurons were hypothesized to be sensitive to stretch
based on the presence of undifferentiated processes in each
motor neuron [1]; this sensory feedback has been proposed
to underlie the propagation of dorsoventral bending waves
during locomotion via a feedback loop. Wen et al. [48] used
static and dynamic microfluidic devices in which portions of
the worm were forced to adopt a straight or curved posture
to show that the locomotory bending wave indeed propagates
via proprioceptive feedback. Using NpHR/Halo-mediated
optogenetic inhibition, the authors then showed that this feedback is mediated by B-type cholinergic motor neurons during
forward movement.
Caenorhabditis elegans’ sinusoidal locomotion depends on
smooth contraction and relaxation of its body wall muscles.
For many years, it was thought that unlike vertebrate skeletal
muscle, which exhibits action potentials, C. elegans body wall
muscles have only graded potentials. However, in 2011
researchers reported that worm muscles do in fact have
calcium-dependent action potentials [87], and that synchronization of action potentials depends on gap junction coupling
between muscle cells [88]. The authors used ChR2-mediated
stimulation of excitatory cholinergic and inhibitory GABAergic
motor neurons to generate specific neuromuscular inputs
during electrophysiological recordings from body wall muscles.
Many aversive stimuli, including head touch, induce a
response consisting of a brief reversal followed by a sharp turn
during which the animal forms an ‘omega’ shape. Donnelly
et al. [89] showed that a shift in the balance between ventral
and dorsal GABAergic signalling contributes to the execution
of a sharp omega turn. Optical activation of the DD motor neurons that innervate the dorsal muscles relaxes the dorsal muscles
and induces a ventral body bend. During optical inhibition of the
DD motor neurons, the dorsal muscles hypercontract, leading to
a dorsal bend. This work was the first to dissect in detail the
neural basis of a stereotyped sequential behaviour.
These studies represent important steps towards an integrative understanding of how the locomotory neural circuit
coordinates behaviour.
(c) Other motor neurons
The paired hermaphrodite-specific neurons HSN are located
near the vulva and innervate muscles involved in egg-laying
behaviour. ChR2-mediated stimulation of HSN, which induces
egg laying, has been used to identify roles of potassium
channels in regulating HSN excitability [42,90].
Defecation in C. elegans is a rhythmic behaviour with a
period of roughly 60 s, mediated by the intestinal cells, body
wall muscles and enteric muscles. Mahoney et al. [91] showed
that ChR2-mediated stimulation of GABAergic motor neurons
rescued expulsive-defective mutants. Wang & Sieburth [92]
used a similar protocol to demonstrate that GABA neuron
stimulation rescued expulsion defects in nlp-40 neuropeptide
mutants but not in mutants lacking exp-1, an excitatory
GABA receptor on enteric muscles. These results showed
that rhythmic neuropeptide release from pacemaker cells to
downstream neurons can execute rhythmic behaviours.
7
Phil. Trans. R. Soc. B 370: 20140212
ChR2-mediated stimulation of motor neurons has been used
in analyses of synaptic transmission at the neuromuscular
junction (NMJ). Prior to the advent of optogenetic methods,
NMJ physiology was studied by extracellular stimulation of
the entire VNC during patch clamp recording from muscle
cells. The non-specific nature of the stimulus made it impossible to discern roles for specific motor neuron types.
Optogenetics has made it straightforward to stimulate
specific neuronal types in combination with electrophysiological recording. Thus, optogenetics has added to the
power of C. elegans to elucidate the molecular machinery of
the synapse and synaptic plasticity.
Quantitative behavioural assays found a decrease in worm
body length during stimulation of excitatory neurons, which
induces contraction of body wall muscles (BWMs). Similarly,
stimulation of GABAergic motor neurons induces muscle relaxation, which transiently increases body length. In dissected
worms, electrophysiological recordings monitored the effect
of optogenetic perturbation on neuromuscular currents. Using
these assays, Liewald et al. examined mutants with defects in
presynaptic and postsynaptic machinery [81]. One surprising
result was that stimulation of cholinergic neurons in presynaptic mutants led to contractions with larger amplitude
than wild-type, potentially owing to compensatory effects in
muscle cells. Kittelmann et al. [82] conducted a study of synaptic
recovery after hyperstimulation of motor neurons (ChR2mediated stimulation until paralysis occurred). Analysis
revealed different time scales of recovery ranging from 8 to
60 s, which were related to endocytosis, scission and regeneration of synaptic vesicles. Optogenetic stimulation has also
been used to study acetylcholine [83] and GABA receptors [84].
Weissenberger et al. [31] used PACa to increase the synaptic output of C. elegans motor neurons, causing an increased
velocity of forward locomotion. The authors highlight this
as an advantage of PACa over ChR2-mediated stimulation:
whereas illumination of a ChR2-expressing cell causes it to
be stimulated, illumination of PACa-expressing cell may
cause an increase in the neuron’s intrinsic output, and may
therefore produce a more physiological output.
Optogenetics has been used to investigate signalling pathways that regulate synaptic plasticity. Jensen et al. [85] optically
stimulated excitatory motor neurons in the VNC using ChR2 in
combination with electrophysiological analysis of muscles and
imaging fluorescent-tagged acetylcholine receptors. The
authors demonstrated activity-dependent synaptic plasticity
at the NMJ and showed that it depends on acetylcholine translocation mediated by Wnt signalling. These results showed that
Wnt signalling regulates the strength of synaptic signalling.
Hoerndii et al. [86] considered mechanisms of synaptic plasticity in AMPA receptors. The authors showed that repetitive
optogenetic stimulation of selected synapses changed the localization of GFP-tagged AMPA receptors in a manner dependent
on UNC-43, the homologue of Caþ2/calmodulin-dependent
protein kinase II (CaMKII), an important mediator of learning
and memory in vertebrates.
(b) Locomotory circuit function
rstb.royalsocietypublishing.org
GABAergic neurons [47]. The majority of motor neurons are
located in the ventral nerve cord (VNC) and innervate the
body wall muscles. Other motor neurons include those controlling head movements, pharyngeal muscles, defecation
and the egg-laying musculature.
Downloaded from http://rstb.royalsocietypublishing.org/ on June 17, 2017
7. Muscles
So far we have considered only the somatic nervous system of
C. elegans. Optogenetics has also been very fruitful in analyses of the much smaller nervous system of the pharynx,
which is responsible for feeding behaviour.
Trojanowski et al. [40] examined the pathways for excitation of the pharyngeal muscle. Previous work using laser
ablation [94] demonstrated that the motor neuron MC is the
only pharyngeal neuron required for rapid pumping. Using
a system for cell-specific optogenetic illumination [41], the
authors showed that not only the MC but also the M2 and
M4 neurons directly stimulate pharyngeal pumping, and
that the MC neurons act via both nicotinic and muscarinic
receptors [40]. In addition, the authors showed that the I1 interneuron stimulates pharyngeal pumping via both MC and M2.
Complementary experiments using optical inhibition via Mac
yielded opposite results, showing that the identified roles of
these neurons are physiological. Inhibition of pharyngeal
pumping during I1 inhibition is surprising, since laser ablation
of I1 has no effect on pumping rate in the presence of bacterial
food [94,95], highlighting the limitations of laser ablation
approaches for understanding circuit function. These results
showed how degenerate neural pathways, in which multiple
elements can perform the same function, regulate behaviour
in a simple model system.
Dillon et al. [96] used ChR2-mediated excitation of glutamatergic pharyngeal neurons to investigate the function of
metabotropic glutamate receptors (mGluRs). The authors
found that glutamate receptors are required for presynaptic
modulation of pharyngeal behaviour and electrical activity.
These findings highlight the essential role of mGluRs in regulating complex behaviours in C. elegans, as they do in vertebrates.
In an analysis of food-dependent locomotory states, Flavell
et al. [75] showed that ChR2-mediated stimulation of the serotonergic pharyngeal sensory/interneuron NSM induced
dwelling behaviour (characterized by a high turn rate and
low velocity) and produced an effect mimicking the presence
Optogenetic analysis has become an indispensable tool for
dissecting the neural basis of behaviour in C. elegans.
Researchers are now able to control individual cell types
from sensory neurons to interneurons to motor neurons to
muscle cells, and quantify the effects of raising or lowering
the activity of individual cells on intact circuits and behaviour. The temporal precision and cellular resolution of
optogenetics in C. elegans have allowed the dissection of a
variety of behaviourally relevant computations in the
nematode nervous system. The rapid expansion of the optogenetics toolkit—more light-sensitive reagents with different
spectral and temporal characteristics—is certain to augment
the utility of optogenetics in worm neuroscience.
As with any experimental method, the limitations and
caveats of optogenetics must be understood to properly
design or interpret experiments. Because the degree of optically mediated excitation or inhibition in any particular cell
is unknown, it is difficult to know how any particular
manipulation compares to physiologically relevant patterns
of activity. Optogenetic manipulations must be coupled to
other methods in C. elegans, such as calcium imaging, genetic
manipulation and quantitative behavioural analysis, to properly gauge their effects. Manipulating neuronal activity with
light, while perhaps minimally invasive, is not non-invasive.
Any transgenic manipulation can disrupt the properties of a
cell in unpredictable ways, and intrinsic light sensitivity may
be compounded with the optogenetic perturbation.
In most cases, optogenetics provides new ways to test models
of circuit function. Because optogenetic perturbations will rarely
recapitulate the normal activity patterns of circuits, they must be
interpreted with care. That said, optogenetics often provides the
most convenient and direct way of raising or lowering the
activity level of any cell type to assess their functional role. Optogenetics is an essential part of the toolbox throughout C. elegans
neuroscience, and its importance will only grow as these tools
become better understood and more widely used.
Competing interests. We declare we have no competing interests.
Funding. C.F.-Y. is supported by the Alfred P. Sloan Foundation, Ellison
Medical Foundation, and the National Institutes of Health. M.J.A. is
supported by the National Institutes of Health (GM084491). A.D.T.S.
is supported by the National Institutes of Health, National Science
Foundation and the Human Frontier Science Program.
Acknowledgements. The authors thank Nicholas Trojanowski and Ni Ji
for helpful comments.
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